ARK5 enhances cell survival associated with mitochondrial morphological dynamics from fusion to fission in human multiple myeloma cells.

5' adenosine monophosphate-activated protein kinase-related kinase 5 (ARK5) is involved in mitochondrial ATP production and associated with poor prognosis of multiple myeloma (MM). However, the molecular mechanisms of ARK5 in MM remain largely unknown. This study examined the pathogenic role of ARK5 in mitochondria by using genetically modified isogenic cell clones with or without ARK5 in human myeloma cell lines, KMS-11 and Sachi, which overexpress ARK5. The biallelic knockout of ARK5 (ARK5-KO) inhibited cell proliferation, colony formation, and migration with increased apoptosis. Mitochondrial fusion was enhanced in ARK5-KO cells, unlike in ARK5 wild-type (ARK5-WT) cells, which exhibited increased mitochondrial fission. Furthermore, ARK5-KO cells demonstrated a lower phosphorylated dynamin-related protein 1 at serine 616, higher protein expression of mitofusin-1 (MFN1) and MFN2, optic atrophy 1 with a lower level of ATP, and higher levels of lactate and reactive oxygen species than ARK5-WT cells. Our findings suggest that ARK5-enhanced myeloma cells can survive associated mitochondrial fission and activity. This study first revealed the relationship between ARK5 and mitochondrial morphological dynamics. Thus, our outcomes show novel aspects of mitochondrial biology of ARK5, which can afford a more advanced treatment approach for unfavorable MM expressing ARK5.

In silico identification and functional prediction of differentially expressed genes in South Asian populations associated with type 2 diabetes.

Type 2 diabetes (T2D) is one of the major metabolic disorders in humans caused by hyperglycemia and insulin resistance syndrome. Although significant genetic effects on T2D pathogenesis are experimentally proved, the molecular mechanism of T2D in South Asian Populations (SAPs) is still limited. Hence, the current research analyzed two Gene Expression Omnibus (GEO) and 17 Genome-Wide Association Studies (GWAS) datasets associated with T2D in SAP to identify DEGs (differentially expressed genes). The identified DEGs were further analyzed to explore the molecular mechanism of T2D pathogenesis following a series of bioinformatics approaches. Following PPI (Protein-Protein Interaction), 867 potential DEGs and nine hub genes were identified that might play significant roles in T2D pathogenesis. Interestingly, CTNNB1 and RUNX2 hub genes were found to be unique for T2D pathogenesis in SAPs. Then, the GO (Gene Ontology) showed the potential biological, molecular, and cellular functions of the DEGs. The target genes also interacted with different pathways of T2D pathogenesis. In fact, 118 genes (including HNF1A and TCF7L2 hub genes) were directly associated with T2D pathogenesis. Indeed, eight key miRNAs among 2582 significantly interacted with the target genes. Even 64 genes were downregulated by 367 FDA-approved drugs. Interestingly, 11 genes showed a wide range (9-43) of drug specificity. Hence, the identified DEGs may guide to elucidate the molecular mechanism of T2D pathogenesis in SAPs. Therefore, integrating the research findings of the potential roles of DEGs and candidate drug-mediated downregulation of marker genes, future drugs or treatments could be developed to treat T2D in SAPs.

Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene.

CRISPR/Cas9 is a powerful genome editing system that has remarkably facilitated gene knockout and targeted knock-in. To accelerate the practical use of CRISPR/Cas9, however, it remains crucial to improve the efficiency, precision, and specificity of genome editing, particularly targeted knock-in, achieved with this system. To improve genome editing efficiency, researchers should first have a molecular assay that allows sensitive monitoring of genome editing events with simple procedures. In the current study, we demonstrate that genome editing events occurring in L1CAM, an X-chromosome gene encoding a cell surface protein, can be readily monitored using flow cytometry (FCM) in multiple human cell lines including neuroblastoma cell lines. The abrogation of L1CAM was efficiently achieved using Cas9 nucleases which disrupt exons encoding the L1CAM extracellular domain, and was easily detected by FCM using anti-L1CAM antibodies. Notably, L1CAM-abrogated cells could be quantified by FCM in four days after transfection with a Cas9 nuclease, which is much faster than an established assay based on the PIGA gene. In addition, the L1CAM-based assay allowed us to measure the efficiency of targeted knock-in (correction of L1CAM mutations) accomplished through different strategies, including a Cas9 nuclease-mediated method, tandem paired nicking, and prime editing. Our L1CAM-based assay using FCM enables rapid and sensitive quantification of genome editing efficiencies and will thereby help researchers improve genome editing technologies.

Quality attributes of the developed banana flour: Effects of drying methods.

The study aims to investigate the effects of different drying methods on the changes in functional properties, physicochemical composition, bioactive compounds, antioxidant activity, sensory attributes, and microstructural quality of the banana flours. Two local banana cultivars, Mehersagar and Sabri, were dried to produce flour using four distinct drying methods: freeze drying (FD), cabinet drying (CD), microwave oven drying (MOD), and forced air oven drying (FOD). The functional properties of the developed banana flours were observed where the findings were as water holding capacity (0.93 ± 0.06-2.74 ± 0.04 g water/g dry sample), oil absorption capacity (0.87 ± 0.06-2.22 ± 0.10 g oil/g dry sample), swelling capacity (4.62 ± 0.02-5.05 ± 0.03 g paste/g dry sample), bulk density (0.54 ± 0.04-0.81 ± 0.02 g/ml), tapped density (0.62 ± 0.04-0.93 ± 0.03 g/ml) and Carr's Index (9.38 ± 0.47-13.58 ± 0.43%). Freeze-dried Mehersagar cultivar's flour showed the leading functional properties with good flowability and cohesiveness. The physicochemical parameters of the flours also revealed significant differences (p < 0.05) in lightness (L*) (50.51 ± 1.49-72.21 ± 1.05), moisture content (3.96 ± 0.09-7.74 ± 0.13%), protein (2.72 ± 0.07-3.93 ± 0.06%), crude fat (0.11 ± 0.01-0.36 ± 0.04%), crude fiber (0.64 ± 0.03-1.22 ± 0.03%), carbohydrate (84.15 ± 0.24-88.26 ± 0.15%) and energy content (354.25 ± 0.57-370.02 ± 0.39 kcal/g). Total flavonoid content (21.44 ± 0.04-34.34 ± 0.03 mgQE/100g) and phenolic content (29.91 ± 0.01-71.46 ± 0.03 mgGAE/100g) was observed, while the highest retention of bioactive compounds was exhibited in Mehersagar cultivar's flour. In terms of appearance, fineness, taste, flavor, color, and overall acceptability, the dried banana flour of both the cultivars obtained from freeze-dried scored overall acceptability 8.04 ± 0.02 and 7.92 ± 0.17, respectively. The scanning electron microscopy analysis of the microstructure of flour granules from each sample revealed a diverse morphological configuration in particle size and shape. According to the findings of this study, the freeze-drying technology is superior to others, and the Mehersagar banana cultivar is more satisfactory in terms of quality characteristics. Moreover, the quality parameters of banana flour may facilitate the formulation of different flour-based gluten-free baked products and food supplements.

Influence of foliar spray and post-harvest treatments on head yield, shelf-life, and physicochemical qualities of broccoli.

Rapid senescence is the key factor in the deterioration of post-harvest shelf-life in broccoli heads. This study evaluates the head yield and its related traits, and physicochemical attributes of broccoli under four foliar sprays of mineral nutrients (B, Zn, Mo, and B + Zn + Mo) with control. The interaction effects of shelf-life and physicochemical attributes of broccoli for these five pre-harvest and five post-harvest storage treatments (LDP bag, HDP vacuum pack, 2% eggshell powder solution, 2% ascorbic acid, and control) both at cold storage and room temperature were evaluated with three replications. The significantly higher marketable head yield of 28.02 t ha-1, maximum gross return [(Bangladesh Taka (BDT 420300 ha-1)], net return (BDT 30565 ha-1), and maximum benefit-cost ratio (BCR) of 3.67 were obtained from the pre-harvest foliar application of B + Zn + Mo in broccoli. Pre-harvest foliar spray of combined nutrient B + Zn + Mo and post-harvest treatment high-density polyethylene (HDP, 15 µm) vacuum packaging efficiently improve post-harvest physicochemical attributes, viz., compactness, green color, texture, carbohydrates, fats, energy, antioxidants, vitamin C, and total phenols in broccoli head compared to the rest of the treatment combinations. In addition, this treatment combination also confirmed a maximum shelf-life of 24.55 days at cold storage [relative humidity (RH) 90-95% and 4°C] and 7.05 days at room temperature (RH 60-65% and 14-22°C) compared to the rest of the treatment combinations. Therefore, we recommend a pre-harvest foliar spray of combined nutrient elements B + Zn + Mo and an HDP (15 µm) vacuum post-harvest packaging for the maximum benefits for both farmers and consumers to get the best head yield, anticipated physicochemical attributes, and maximum shelf-life of broccoli.

The anti-TLR4 monoclonal antibody Sa15-21 enhances inflammatory cytokine production in LPS-stimulated macrophages.

Sa15-21, a monoclonal antibody against mouse Toll-like receptor (TLR) 4, can protect mice from lipopolysaccharide (LPS)/D-galactosamine-induced acute lethal hepatitis. Herein, we investigated the molecular mechanisms underlying Sa15-21-mediated regulation of TLR4 signaling in macrophages. Results showed that Sa15-21 enhanced the production of proinflammatory cytokines and attenuated the production of anti-inflammatory cytokines in LPS-stimulated macrophages. Western blotting analysis revealed that Sa15-21 pretreatment had no effect on NF-κB and MAPK signaling in LPS-stimulated macrophages; however, Sa15-21 treatment alone led to a weak and delayed activation of NF-κB and MAPK signaling without any effect on proinflammatory cytokine production. By contrast, Sa15-21 failed to induce the activation of interferon regulatory factor 3. Taken together, our results indicate that Sa15-21 sensitizes macrophages to facilitate the inflammatory response via TLR signaling.

Phylogenetic analysis and characterization of arsenic (As) transforming bacterial marker proteins following isolation of As-tolerant indigenous bacteria.

Marker proteins play a significant role in bacterial arsenic (As) transformation. Phylogenetic analysis and three-dimensional (3D) characteristics of As transforming bacterial marker proteins guide the evolutionary origin and As transforming potential of the species. Indeed, As-tolerant bacteria also show a significant level of As transformation. Hence, characterization of As transforming bacterial marker proteins, isolation of As transforming bacteria, and proper integration of the findings may guide to elucidate how bacteria transform As. Therefore, phylogenetic analysis and 3D characterization of As transforming bacterial marker protein following isolation of potential indigenous As-tolerant indigenous bacteria were done to explore the mechanism of bacterial As transformation. Phylogenetic analysis of ten As transforming marker proteins (arsA, arsB, arsC, arsD, arsR, aioA, arrA, aioB, acr1, and acr3) in 20 potential bacterial genomes (except 19 for the acr3) were studied. Some bacterial genomes featured up to five marker proteins, and therefore, 3D characteristics of the marker proteins were analyzed in those genomes having three-to-five marker proteins. In phylogeny, species in close clades represent their phylogenetic resemblances and may have similar functions. P. aeruginosa, E. coli, and K. pneumonia were found to be more effective due to having the highest number (five) of marker proteins. In 3D protein modeling, most of the marker proteins were found to be active. Among 19 indigenous bacterial isolates, multiple isolates showed tolerance up to 50 mM As(III) and 250 mM As(V), which may potentially transform a significant quantities of As. Hence, integration of the results of phylogenetic analysis, 3D protein characteristics, and As tolerance in the bacterial isolates could guide to explore the mechanism of how bacteria transform As at cellular and molecular levels.

Cell surface expression of human RP105 depends on N-glycosylation of MD-1.

For its cell surface expression, radioprotective 105 (RP105) - an orphan Toll-like receptor - must form a complex with a soluble glycoprotein called myeloid differentiation 1 (MD-1). The number of RP105-negative cells is significantly increased in patients with systemic lupus erythematosus (SLE); however, to elucidate the mechanism underlying this increase, how RP105 is expressed on the cell surface depending on MD-1 should be investigated. We demonstrated that RP105 exhibits two forms depending on MD-1 and its two N-glycosylation sites, N96 and N156. Cell surface expression of RP105 decreased in the presence of mutant MD-1 (N96Q/N156Q). Nonglycosylated MD-1 decreased the de novo cell surface expression of RP105 but not pre-expressed RP105. Thus, the N-glycans of MD-1 may represent targets for SLE therapy.

Broadly Neutralizing Antibodies for Influenza: Passive Immunotherapy and Intranasal Vaccination.

Influenza viruses cause annual epidemics and occasional pandemics. The high diversity of viral envelope proteins permits viruses to escape host immunity. Therefore, the development of a universal vaccine and broadly neutralizing antibodies (bnAbs) is essential for controlling various mutant viruses. Here, we review some potentially valuable bnAbs for influenza; one is a novel passive immunotherapy using a variable domain of heavy chain-only antibody (VHH), and the other is polymeric immunoglobulin A (pIgA) induced by intranasal vaccination. Recently, it was reported that a tetravalent multidomain antibody (MDAb) was developed by genetic fusion of four VHHs, which are bnAbs against the influenza A or B viruses. The transfer of a gene encoding the MDAb-Fc fusion protein provided cross-protection against both influenza A and B viruses in vivo. An intranasal universal influenza vaccine, which can induce neutralizing pIgAs in the upper respiratory tract, is currently undergoing clinical studies. A recent study has revealed that tetrameric IgAs formed in nasal mucosa are more broadly protective against influenza than the monomeric and dimeric forms. These broadly neutralizing antibodies have high potential to control the currently circulating influenza virus.

A Novel Gene Delivery Vector of Agonistic Anti-Radioprotective 105 Expressed on Cell Membranes Shows Adjuvant Effect for DNA Immunization Against Influenza.

Radioprotective 105 (RP105) (also termed CD180) is an orphan and unconventional Toll-like receptor (TLR) that lacks an intracellular signaling domain. The agonistic anti-RP105 monoclonal antibody (mAb) can cross-link RP105 on B cells, resulting in the proliferation and activation of B cells. Anti-RP105 mAb also has a potent adjuvant effect, providing higher levels of antigen-specific antibodies compared to alum. However, adjuvanticity is required for the covalent link between anti-RP105 mAb and the antigen. This is a possible obstacle to immunization due to the link between anti-RP105 mAb and some antigens, especially multi-transmembrane proteins. We have previously succeeded in inducing rapid and potent recombinant mAbs in mice using antibody gene-based delivery. To simplify the covalent link between anti-RP105 mAb and antigens, we generated genetic constructs of recombinant anti-RP105 mAb (αRP105) bound to the transmembrane domain of the IgG-B cell receptor (TM) (αRP105-TM), which could enable the anti-RP105 mAb to link the antigen via the cell membrane. We confirmed the expression of αRP105-TM and the antigen hemagglutinin, which is a membrane protein of the influenza virus, on the same cell. We also found that αRP105-TM could activate splenic B cells, including both mature and immature cells, depending on the cell surface RP105 in vitro. To evaluate the adjuvanticity of αRP105-TM, we conducted DNA immunization in mice with the plasmids encoding αRP105-TM and hemagglutinin, followed by challenge with an infection of a lethal dose of an influenza virus. We then obtained partially but significantly hemagglutinin-specific antibodies and observed protective effects against a lethal dose of influenza virus infection. The current αRP105-TM might provide adjuvanticity for a vaccine via a simple preparation of the expression plasmids encoding αRP105-TM and of that encoding the target antigen.

Receptor-destroying enzyme (RDE) from Vibrio cholerae modulates IgE activity and reduces the initiation of anaphylaxis.

IgE plays a key role in allergies by binding to allergens and then sensitizing mast cells through the Fc receptor, resulting in the secretion of proinflammatory mediators. Therefore, IgE is a major target for managing allergies. Previous studies have reported that oligomannose on IgE can be a potential target to inhibit allergic responses. However, enzymes that can modulate IgE activity are not yet known. Here, we found that the commercial receptor-destroying enzyme (RDE) (II) from Vibrio cholerae culture fluid specifically modulates IgE, but not IgG, and prevents the initiation of anaphylaxis. RDE (II)-treated IgE cannot access its binding site on bone marrow-derived mast cells, resulting in reduced release of histamine and cytokines. We also noted that RDE (II)-treated IgE could not induce passive cutaneous anaphylaxis in mouse ears. Taken together, we concluded that RDE (II) modulates the IgE structure and renders it unable to mediate allergic responses. To reveal the mechanism by which RDE (II) interferes with IgE activity, we performed lectin microarray analysis to unravel the relationship between IgE modulation and glycosylation. We observed that RDE (II) treatment significantly reduced the binding of IgE to Lycopersicon esculentum lectin, which recognizes poly-N-acetylglucosamine and poly-N-acetyllactosamine. These results suggest that RDE (II) specifically modulates branched glycans on IgE, thereby interfering with its ability to induce allergic responses. Our findings may provide a basis for the development of drugs to inhibit IgE activity in allergies.